Figure 10: The contribution of gut environmental stimuli to the induction of CD4⁺ IELs.

(a) Six-week-old 2D2 mice were orally treated with distilled water (control) or a mixture of antibiotics (Abx) (n=7 per group). Two weeks later, IELs were collected and analysed for absolute numbers of 2D2-IEL cells. Each dot represents an individual mouse. Data are pooled from two experiments (mean and s.e.m.). (b) The microarray gene expression analysis showed a pattern of higher expression in WT-CD4⁺CD8α⁻ CD25⁻IEL (WT-Gut) and CD4⁺CD25⁻2D2-IEL-T_HIGH cells (2D2-Gut) compared with WT-CD4⁺CD25⁻ spleen-T cells (WT-Sp) and CD4⁺CD25⁻2D2-spleen-T cells (2D2-Sp). The log₂ expression values of AHR signalling pathway-related genes are shown in the heatmap. Each column represents a single sample (n=3 per group) and each row represents a single gene (38 genes). The result of the ingenuity pathway analysis is shown below (see Method). (c) A standard diet (control), synthetic purified diet (AIN76A) or synthetic diet supplemented with I3C (AIN76A+I3C) was fed to WT mice. After 1 month, IELs were obtained and the absolute numbers of CD4⁺CD8α⁻TCRβ⁺ IELs were analysed (n=10 per group). Each dot represents an individual animal. Pooled data from two experiments are shown (mean and s.e.m.). (a) *P<0.05 by unpaired t-test. (c) *P<0.05 by one-way analysis of variance with Bonferroni’s post-test.